The in vitro susceptibilities of 36 clinical isolates of Madurella mycetomatis, the prime agent of eumycetoma in Africa, to ketoconazole, itraconazole, fluconazole, voriconazole, amphotericin B, and flucytosine were determined by the Sensititre YeastOne system. This system appeared to be a rapid and easy test, and by use of hyphal suspensions it generated results comparable to those of a modified NCCLS method. After 10 days of incubation, the antifungal activities of ketoconazole (MIC at which 90% of isolates were inhibited [MIC90], 0.125 microg/ml), itraconazole (MIC90, 0.064 microg/ml), and voriconazole (MIC90, 0.125 microg/ml) appeared superior to those of fluconazole (MIC90, 128 microg/ml) and amphotericin B (MIC90, 1 microg/ml), with MICs in the clinically relevant range. All isolates were resistant to flucytosine (all MICs above 64 microg/ml). Based on the relatively broad range of MICs obtained for the antifungal agents, routine testing of M. mycetomatis isolates for susceptibility to antifungal agents seems to be relevant to adequate therapeutic management.